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Crystal structure of tak1-tab1Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Enzyme (e.g., Ligases (6. ), Etc.), Proenzyme; Compositions Thereof; Process For Preparing, Activating, Inhibiting, Separating, Or Purifying Enzymes, Transferase Other Than Ribonuclease (2.), Transferring Phosphorus Containing Group (e.g., Kineases, Etc.(2.7))Crystal structure of tak1-tab1 description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070178573, Crystal structure of tak1-tab1. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This non-provisional patent application claims priority to U.S. Provisional Application No. 60/655,606, filed Feb. 23, 2005, which is incorporated by reference in its entirety TECHNICAL FIELD OF THE INVENTION [0002] The present invention relates to the design of crystallisable transforming growth factor-beta-activated kinase 1 (TAK1) and TAK1 binding protein (TAB1) complexes and the X-ray analysis of crystalline molecules or molecular complexes of this TAK1--TAB1 chimera. The present invention provides a chimera of TAK1 and a region of the TAK1 activating domain of its protein activator TAB1. The present invention also provides for the first time the crystal structure of a TAK1--TAB1 chimera protein bound to adenosine and TAK1--TAB1 bound to a potent ATP-competitive inhibitor. The present invention also provides crystalline molecules or molecular complexes that comprise binding pockets of TAK1 kinase (TAK1) and/or its structural homologues, the structure of these molecules or molecular complexes. The present invention further provides crystals of TAK1--TAB1 complexed with adenosine and methods for producing these crystals. This invention also relates to a general strategy for the design of crystallisable protein kinases based on both sequence and structural alignments of related protein kinases with close homology that have previously been crystallized in the literature. This invention also relates to crystallizable compositions from which the protein-ligand complexes may be obtained. The present invention also relates to a data storage medium encoded with the structural coordinates of molecules and molecular complexes that comprise the ATP-binding pockets and TAB1-binding pockets of TAK1 or their structural homologues. The present invention also relates to a computer comprising such data storage material. The computer may generate a three-dimensional structure or graphical three-dimensional representation of such molecules or molecular complexes. This invention also relates to methods of using the structure coordinates to solve the structure of homologous proteins or protein complexes. This invention also relates to computational methods of using structure coordinates of the TAK1 complex(es) to screen for and design compounds, including inhibitory compounds and antibodies, that interact with TAK1, TAB1 or homologues thereof. BACKGROUND OF THE INVENTION [0003] The search for new therapeutic agents has been greatly aided in recent years by a better understanding of the structure of enzymes and other biomolecules associated with diseases. One important class of enzymes that has been the subject of extensive study is protein kinases. [0004] Protein kinases constitute a large family of structurally related enzymes that are responsible for the control of a variety of signal transduction processes within the cell. (See, Hardie, G. and Hanks, S. The Protein Kinase Facts Book, I and II, Academic Press, San Diego, Calif.: 1995). Protein kinases are thought to have evolved from a common ancestral gene due to the conservation of their structure and catalytic function. Almost all kinases contain a similar 250-300 amino acid catalytic domain. The kinases may be categorized into families by the substrates they phosphorylate (e.g., protein-tyrosine, protein-serine/threonine, lipids, etc.). Sequence motifs have been identified that generally correspond to each of these kinase families (See, for example, Hanks, S. K., Hunter, T., FASEB J., 9:576-596 (1995); Knighton et al., Science, 253:407-414 (1991); Hiles et al., Cell, 70:419-429 (1992); Kunz et al., Cell, 73:585-596 (1993); Garcia-Bustos et al., EMBO J., 13:2352-2361 (1994)). [0005] In general, protein kinases mediate intracellular signaling by effecting a phosphoryl transfer from a nucleoside triphosphate to a protein acceptor that is involved in a signaling pathway. These phosphorylation events act as molecular on/off switches that can modulate or regulate the target protein biological function. These phosphorylation events are ultimately triggered in response to a variety of extracellular and other stimuli. Examples of such stimuli include environmental and chemical stress signals (e.g., osmotic shock, heat shock, ultraviolet radiation, bacterial endotoxin, and H.sub.2O.sub.2), cytokines (e.g., interleukin-1 (IL-1) and tumor necrosis factor .alpha. (TNF-.alpha.)), and growth factors (e.g., granulocyte macrophage-colony-stimulating factor (GM-CSF), and fibroblast growth factor (FGF)). An extracellular stimulus may affect one or more cellular responses related to cell growth, migration, differentiation, secretion of hormones, activation of transcription factors, muscle contraction, glucose metabolism, control of protein synthesis, and regulation of the cell cycle. [0006] Many diseases are associated with abnormal cellular responses triggered by protein kinase-mediated events as described above. These diseases include, but are not limited to, autoimmune diseases, inflammatory diseases, bone diseases, metabolic diseases, neurological and neurodegenerative diseases, cancer, cardiovascular diseases, allergies and asthma, Alzheimer's disease, and hormone-related diseases. Accordingly, there has been a substantial effort in medicinal chemistry to find protein kinase inhibitors that are effective as therapeutic agents. [0007] Among medically important serine/threonine kinases is the family of mitogen-activated protein kinases (MAPKs), which have been shown to function in a wide variety of biological processes (Davis D. J. Trends in Biochem Sci. 19 470-473 (1994); Su B. & Karin M Curr. Opin. Immunol 8 402-411 (1996); Treisman R. Curr. Opin. Cell Biol. 8 205-215 (1996)). MAPKs are activated by phosphorylation on specific tyrosine and threonine residues by MAPK kinases (MAPKKs), which are in turn activated by phosphorylation on serine and serine/threonine residues by MAPKK kinases (MAPKKKs). The MAPKKK family comprises several members including MEKK1, MEKK3, NIK and ASK1 and Raf. Different mechanisms are involved in the activation of MAPKKKs in response to a variety of extracellular stimuli including cytokines, growth factors and environmental stresses (refs). [0008] Transforming growth factor-.beta. (TGF-.beta.)-activated kinase 1 (TAK1) is a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family and has been shown to play critical roles in signaling pathways stimulated by transforming growth factor-.beta., interleukin-1 (IL-1), tumor necrosis factor-.alpha. (TNF-.alpha.), lipopolysaccharide, receptor activator of NF-.kappa.B ligand where it regulates osteoclast differentiation and activation, and IL-8 (Yamaguchi K et al. Science 270 2008-11 (1995); Ninomiya-Tsuji J et al. Nature 398 252-256 (1999); Sakurai H. et al. J. Biol. Chem 274 10641-10648 (1999); Irie T. et al. FEBS Lett. 467 160-164 (2000); Lee J. et al. J. Leukoc Biol. 68 909-915 (2000); Mizukami J et al. Mol. Cell. Biol. 22 992-1000 (2002); Wald D. et al. J. Immunol. 31 3747-3754 (2002)). TAK1 regulates both the c-Jun N-terminal kinase (JNK) and p38 MAPK cascades in which it phosphorylates MAPK kinases MKK4 and MKK3/6, respectively (Wang W. et al. J. Biol. Chem. 272 22771-22775 (1997); Moriguchi T. et al. J. Biol. Chem. 271 13675-13679 (1996)). NF-kB factors regulate expression of a variety of genes involved in apoptosis, cell cycle, transformation, immune response, and cell adhesion (Barkett M and Gilmore T D. Oncogene, 18, 6910-6924 (1999). TAK1 regulates the I.kappa.B kinase (IKK) signaling pathways, leading to the activation of transcription factors AP-1 and NF-.kappa.B (Ninomiya-Tsuji J et al. Nature 398 252-256 (1999); Sakurai H. et al. J. Biol. Chem 274 10641-10648 (1999); Takaesu G. et al. J. Mol. Biol. 326 110-115 (2003)). In early embryos of the amphibian Xenopus, TAK1 also participates in mesoderm induction and patterning mediated by bone morphogenetic protein (BMP), which is another transforming growth factor .beta. family ligand (Shibuya H. et al. EMBO J. 17 1019-1028 (1998)). In addition, TAK1 is a negative regulator of the Wnt signaling pathway, in which TAK1 down-regulates transcription regulation mediated by a complex of .beta.-catenin and T-cell factor/lymphoid enhancer factor (Meneghini M. D. et al. Nature 399 793-797 (1999); Ishitani T. et al. Nature 399 798-802 (1999)). The role of TAK1 in TNF-.alpha. and IL-1.beta.-induced signaling events is evident from TAK1 RNAi experiments in mammalian cells (Takaesu G. et al. J. Mol. Biol. 326 105-115 (2003)) in which IL-1 and TNF-.alpha. induced NF-.kappa.B and MAPK activation were both inhibited. Over-expression of kinase dead TAK1 inhibits IL-1 and TNK-induced activation of both JNK/p38 and NF-kB (Ninomiya-Tsuji J et al. Nature 398 252-256 (1999); Sakurai H. et al. J. Biol. Chem 274 10641-10648 (1999)). TAK1-/- mouse embryonic fibroblasts have diminished IL-1-induced signaling and are embryonic lethal (E11.5) (S. Akira, personal communication). In adult mouse, TAK1 is activated in the myocardium after pressure overload. Expression of constitutively-active TAK1 in myocardium induced myocardial hypertrophy and heart failure in transgenic mice (Zhang D. et al. Nature Med. 6 556-563 (2000)). [0009] TAK1 is activated by the TAK1 binding protein (TAB1) (Shibuya H et al. Science 272 1179-1182 (1996)) via an association with the N-terminal kinase domain of TAK1. It has been reported that the C-terminal 68 amino acids of TAB1 is sufficient for the association and activation of TAK1 (Shibuya H et al. Science 272 1179-1182 (1996)). However, more recent work indicates that the minimum TAB1 segment required includes only residues 480-495 (Ono K. et al. J. Biol. Chem. 276 24396-24400 (2001); Sakurai H. et al. FEBS Lett 474 141-145 (2000)). Deletion mutants of TAB1 show that the aromatic Phe484 residue is critical for TAK1 binding (Ono K. et al. J. Biol. Chem. 276 24396-24400 (2001)). Autophosphorylation of threonine/serine residues in the kinase activation loop are necessary for TAB1-induced TAK1 activation (Sakurai H. et al. FEBS Lett 474 141-145 (2000); Kishimoto K. et al. J. Biol. Chem. 275 7359-7364 (2000)), Ser192 appears as the most likely candidate since a Ser192Ala mutation shows no kinase activity (Kishimoto K. et al. J. Biol. Chem. 275 7359-7364 (2000)). [0010] Since TAK1 is a key molecule in the pro-inflammatory NF-.kappa.B signaling pathway a TAK1 inhibitor would be effective in diseases associated with inflammation and tissue destruction such as rheumatoid arthritis and inflammatory bowel disease (Crohn's), as well as in cellular processes such as stress responses, apoptosis, proliferation and differentiation. Various pro-inflammatory cytokines and endotoxins trigger the kinase activity of endogenous TAK1 (Ninomiya-Tsuji J et al. Nature 398 252-256 (1999); Irie T et al. FEBS Lett. 467 160-164 (2000); Sakurai H. et al. J. Biol. Chem. 274 10641-10648 (1999)) and the Drosophila homolog of TAK1 was recently identified as an essential molecule for host defense signaling in Drosophila (Vidal S. et al. Genes Dev. 15 1900-1912 (1999)). A natural inhibitor of TAK1, 5Z-7-oxozeaenol, has been identified with an IC50 value of 8 nM. 5Z-7-oxozeaenol has been shown to be selective for TAK1 within the MAPKKK family and relieves inflammation in a picryl chloride-induced ear swelling mouse model (Ninomiya-Tsuji J. et al. J. Biol. Chem. 278 18485 (2003)). [0011] Accordingly, there has been an interest in finding selective inhibitors of TAK1 that are effective as therapeutic agents. A challenge has been to find protein kinase inhibitors that act in a selective manner, targeting only TAK1. Since there are numerous protein kinases that are involved in a variety of cellular responses, non-selective inhibitors may lead to unwanted side effects. In this regard, the three-dimensional structure of the kinase would assist in the rational design of inhibitors. The determination of the amino acid residues in TAK1 binding pockets and the determination of the shape of those binding pockets would allow one to design selective inhibitors that bind favorably to this class of enzymes. The determination of the amino acid residues in TAK1 binding pockets and the determination of the shape of those binding pockets would also allow one to determine the binding of compounds to the binding pockets and to, e.g., design inhibitors that can bind to TAK1. [0012] For example, a general approach to designing inhibitors that are selective for an enzyme target is to determine how a putative inhibitor interacts with the three dimensional structure of the enzyme. For this reason it is useful to obtain the enzyme protein in crystal form and perform X-ray diffraction techniques to determine its three dimensional structure coordinates. If the enzyme is crystallized as a complex with a ligand, one can determine both the shape of the enzyme binding pocket when bound to the ligand, as well as the amino acid residues that are capable of close contact with the ligand. By knowing the shape and amino acid residues in the binding pocket, one may design new ligands that will interact favorably with the enzyme. With such structural information, available computational methods may be used to predict how strong the ligand binding interaction will be. Such methods thus enable the design of inhibitors that bind strongly, as well as selectively to the target enzyme. [0013] Despite the fact that the genes for TAK1 has been isolated and the amino acid sequence of TAK1 is known, no one has described X-ray crystal structural coordinate information of TAK protein. As disclosed herein, such information would be extremely useful in identifying and designing potential inhibitors of the TAK kinase or homologues thereof, which, in turn, could have therapeutic utility. [0014] The structures of several serine/threonine kinases have been solved by X-ray diffraction and analyzed. Specifically, the crystal structures of P38 kinase (Wilson et al., J. Biol. Chem., 271, pp. 27696-27700 (1996)) and MAPKAP Kinase 2 (U.S. Provisional application 60/337,513) have been studied in detail. [0015] To date, no crystal structures of TAK kinase have been reported. Thus the crystal structure of unphosphorylated TAK kinase domain complexes with inhibitors are of great importance for defining the active conformation of TAK kinase. This information is essential for the rational design of selective and potent inhibitors of TAK. SUMMARY OF THE INVENTION [0016] The present invention provides for the first time, crystallizable compositions, crystals, and the crystal structures of a TAK1--inhibitor complex. The TAK1 protein used in these studies corresponds to a single polypeptide chain, which encompasses the complete catalytic kinase domain, amino acids 31 to 303 fused to the C-terminal 36 amino acids of TAB1 (468 to 504). Solving this crystal structure has allowed the applicants to determine the key structural features of TAK1, particularly the shape of its substrate and ATP-binding pockets. [0017] Thus, in one aspect, the present invention provides molecules or molecular complexes comprising all or parts of these binding pockets, or homologues of these binding pockets that have similar three-dimensional shapes. [0018] In another aspect, the present invention further provides crystals of TAK1 complexed with adenosine and methods for producing these crystals. In this embodiment, TAK1 is unphosphorylated. [0019] In a further aspect, the present invention provides crystallizable compositions from which TAK1-ligand complexes may be obtained. [0020] In another aspect, the present invention provides for a general strategy for the design of protein constructs for producing crystallisable kinase domains. Continue reading about Crystal structure of tak1-tab1... Full patent description for Crystal structure of tak1-tab1 Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Crystal structure of tak1-tab1 patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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